Kinetochores are large protein structures assembled on centromeric chromatin that power and regulate chromosome segregation during mitosis. Properly functioning kinetochores are essential for mitotic cell division and for maintaining human health, as defective chromosome segregation is implicated as a causative factor in the formation of birth defects and in the initiation and progression of cancer. Kinetochores are considered the orchestrators of mitosis for several reasons. Kinetochores physically connect mitotic chromosomes to spindle microtubules (MTs) and transduce forces through this connection to congress chromosomes; they regulate the binding strength between chromosomes and MTs so that improper attachments are corrected; and, finally, they ensure that cells do not exit mitosis if chromosomes fail to attach or are incorrectly attached to MTs by regulating the spindle assembly checkpoint (SAC), which monitors kinetochore-MT attachment and prevents anaphase in such unfavorable conditions. The NDC80 complex plays a central role in the generation and regulation of attachments: it is the primary linkage between kinetochores and MTs, and phosphorylation of its Hec1 subunit by the essential Aurora B kinase (ABK) regulates kinetochore-MT attachment strength. The goals of this study are to understand how kinetochore-MT attachments are precisely regulated during mitotic progression and to determine how kinetochore-MT stability is linked to SAC signaling. These issues will be addressed in three Aims. The first Aim will investigate how NDC80-mediated kinetochore-MT attachment is regulated by phosphorylation and dephosphorylation and how the SAC detects stable NDC80-MT connections. Using a combination of in vivo, in vitro, and in silico approaches, the mechanisms governing Hec1 dephosphorylation during mitosis will be determined, the contributions of ABK and non-ABK mitotic kinases to Hec1 phosphorylation will be mapped, and how Hec1 regulates the recruitment and eviction of upstream SAC activators will be determined. In the second Aim, the mechanism for ABK-mediated regulation of kinetochore-MT attachment stability will be addressed by building on exciting preliminary data suggesting that kinetochore substrates, including Hec1, are phosphorylated by a population of ABK recruited specifically to kinetochores, rather than by a population of ABK at inner centromeres. To determine the molecular basis for this activity, cell biological approaches and in vitro reconstitution assays using purified proteins and nucleosome arrays will be employed. In the third Aim, unexplored roles of the kinetochore scaffold protein KNL1 will be investigated. How KNL1 recruits both SAC silencers and activators will be determined, and the functions of recently identified KNL1-interacting proteins will be characterized. In summary, this proposal will address how kinetochore-MT attachments are regulated and monitored during mitosis to ensure accurate chromosome segregation using multiple approaches, including quantitative cell-based high-resolution and super-resolution microscopy, biochemical reconstitution, single- molecule biophysical assays, and mathematical modeling.